Most human pathogens enter via mucosal routes. Yet, there are very few licensed mucosal vaccines. Thus, there is a need for new vaccine technologies or adjuvants that stimulate protective immunity at mucosal sites. However, current mucosal adjuvants have significant limitations, including toxicity. We propose to develop/establish a novel recombinant, adjuvant-free, mucosal vaccine platform. The potential for an adjuvant-free mucosal vaccine platform is demonstrated using a mono [human Fc?RI (hFc?RI)]-specific prototype fusion protein (FP) consisting of pneumococcal surface protein A (PspA) antigen (Ag) targeted to hFc?RI. Specifically, this prototype FP enhances immunogenicity and protection against a mucosal S. pneumoniae (Sp) challenge when administered intranasally (i.n.) to hFc?RI transgenic mice, in the absence of adjuvant. We now propose creating a dual-targeted (multi-functional) FP which: 1) Enhances delivery of vaccine Ag to hFc?RI-expressing Ag presenting cells (APCs) within the nasal-associated lymphoid tissue (NALT), via FcRn mediated transepithelial transport of FP. 2) Further enhances Ag internalization, dendritic cell (DC) maturation, and Ag presentation/T cell activation, via increased hFc?RI crosslinking on APCs within the NALT. Importantly, with the current focus of vaccine research primarily on adjuvant discovery, maximizing the potency of this adjuvant-free vaccine platform will be crucial to changing perceptions regarding the need for adjuvant. To achieve this, we will: Aim 1) Add an FcRn targeting sequence to the prototype (mono-targeted) PspA containing FP, as well as convert the bivalent FP to a trivalent FP. This novel multi-functional FP will then be tested in vitro for its ability to sequentially enhance transepithelial transport of Ag, Ag internalization, DC maturation, and Ag presentation/T cell activation. Aim 2: Optimize FP platform immunogenicity and protective efficacy utilizing the Sp PspA Ag mucosal vaccine model and hFcRn/hFc?RI-expressing mice. Specifically, we will compare bivalent vs. trivalent hFcRI-targeted FP (plus and minus the FcRn targeting component) i.n. The most protective FP will then be selected for further development and will also be tested for immunity and protection against multiple Sp strains versus a licensed Sp vaccine. Aim 3: Utilize an in vitro transwell model of the human nasal tract consisting of a human epithelial cell layer in the top well (representing the nasal lumen) and human immune cells in the lower well (representing the NALT). The ability of the optimized FP to transport Ag from the upper well to the lower well and subsequently enhance human T and B cell responses in the lower well will be examined. Ag-specific antibody responses will be measured by ELISA/ELISPOT. T cell activation will be measured via increased CD25 expression, T cell proliferation, and cytokine production. The proposed studies will be crucial to establishing this novel and innovative (dual-targeted/multi-functional) vaccine platform as a viable adjuvant-free approach for mucosal immunization. Furthermore, its success will fundamentally transform the paradigm for vaccine generation/administration against a wide array of infectious disease agents.